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Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (Comet) assay.
Mutation Research 393 Ž1997. 107–113
Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis ž Comet/ assay. 2. Relationship between DNA migration and alkaline condition Youichi Miyamae a , Kouichi Iwasaki b,1, Naohide Kinae b, Shuji Tsuda c , Michiko Murakami d,2 , Makiko Tanaka d,3, Yu F. Sasaki d,) a
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 1-6, 2-chome, Kashima, Yodogawa-Ku, Osaka 532, Japan Laboratory of Food Hygiene, School of Food and Nutritional Sciences, UniÕersity of Shizuoka, Yada 52-1, Shizuoka 422, Japan c Laboratory of Veterinary Public Health, Faculty of Agriculture, Iwate UniÕersity, Ueda 3-18-8, Morioka, Iwate 020, Japan Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 16-1, Hachinohe, Aomori 039-11, Japan b
d
Received 21 May 1996; revised 11 April 1997; accepted 23 April 1997
Abstract The alkaline condition is an important factor for the alkaline single-cell gel electrophoresis ŽSCG. assay to detect the genotoxic effects of chemicals. In order to understand the relationship between DNA migration and alkaline condition, the effect of 13 model chemical mutagens with different modes of action was evaluated with the alkaline SCG assay under two different alkaline conditions ŽpH 12.1 and 12.6.. CHO cells were sampled just after treatment for 1 h. The X-ray mimetic mutagen BLM increased DNA migration at pH 12.1 and 12.6 and the results were the same at both pH values. Six alkylating mutagens MNU, ENU, MNNG, ENNG, MMS, and EMS and one base adduct inducer 4-NQO induced a dose-dependent response only at pH 12.6. Two DNA crosslinking agents, MMC and DDP, and AMD had negative results. MMC and DDP, however, reduced the positive response of BLM, suggesting that DNA crosslinks could be detected. These results demonstrated that the alkaline condition was important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals. q 1997 Elsevier Science B.V. Keywords: Alkaline single cell gel electrophoresis ŽSCG. assay; Comet assay; DNA lesion; pH; Chemical mutagen
X
X
Abbreviations: MNNG, N-methyl-N -nitro-N-nitrosoguanidine; ENNG, N-ethyl-N -nitro-N-nitrosoguanidine; MNU, methyl nitrosourea; ENU, ethyl nitrosourea; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; 4-nitroquinoline 1-oxide, 4-NQO; MMC, mitomycin C; DDP, cis-diaminodichloroplatin; AMD, actinomycin D; BLM, bleomycin; MX, 3-chloro-4-dichloromethyl-5-hydroxy2Ž5H .-furanone; SSB, DNA single-strand break ) Corresponding author. Tel.rfax: q81 Ž178. 27-7296; e-mail: [email protected] 1 Present address: Water Research Foundation Shizuoka Prefecture, Kita-andoh, Shizuoka 420, Japan. 2 Present address: Faculty of Engineering, The Technology University of Nagaoka, Kamitomioka 1603-1, Nagaoka, Niigata 940-21, Japan. 3 Present address: Tokyo Factory, Nisshinbo Co. Ltd., Nishiarai Sakae 1-18-1, Adachi, Tokyo 123, Japan. 1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 9 1 - 0
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Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
1. Introduction The alkaline single-cell gel electrophoresis ŽSCG. assay is a rapid and sensitive procedure for quantitating DNA lesions in mammalian cells w1,2x. In this assay, cells are embedded in agarose, lysed in an alkaline buffer, and subjected to an electric current. Relaxed and broken DNA fragments stream further from the nucleus than intact DNA, so the extent of DNA damage can be measured by the length of the stream. DNA is denatured and unwound and electrophoresis is conducted under alkaline condition ŽpH ) 12. w1,2x. At pH G 12.6, SSBs develop from DNA lesions called alkali-labile sites w3x. Thus, only SSBs will be detected at pH 12.1, and both SSBs and alkali-labile sites will be detected at pH G 12.6. Since DNA crosslinking lesions which inhibit DNA unwinding inhibit the fragmentation of DNA molecules rather than induce a comet tail, DNA crosslinking lesions as well as SSBs and alkali-labile sites can be detected by the alkaline SCG assay w4,5x. Some kinds of mutagen react DNA with UVandror X-ray mimetic modeŽs. and induce SSBs andror alkali-labile sites. The alkaline condition, therefore, is an important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals. In this study, we questioned the relationship between DNA migration and alkaline condition. We selected the following as model mutagens: the S N -1 type alkylating agents MNNG, ENNG, MNU, and ENU, the S N -2 type alkylating agents MMS and EMS, the base adduct inducers 4-NQO and MX, the crosslinking agents MMC and DDP, the DNA intercalator AMD, the X-ray mimetic mutagen BLM. According to Vijayalaxmi, et al., pH ) 13 would be more useful than pH 12.6 for detecting certain classes of alkali-labile lesions w6x. To understand the relationship between DNA migration and alkaline condition, however, we used the lowest pH ŽpH 12.6. at which alkali-labile sites could be detected. 2. Materials and methods 2.1. Chemicals, cells and medium MNNG, ENNG, MMS, and EMS were obtained from Sigma Chemicals Inc., St. Louis, MO ŽUSA..
MNU and ENU were purchased from Nacalai Tesque, Inc., Kyoto ŽJapan.. 4-NQO was obtained from Wako Pure Chemical Industries, Ltd., Osaka ŽJapan.. MMC and DDP came from Kyowa Hakko Kogyo Ltd., Tokyo ŽJapan., and Nihon Kayaku Industries, Ltd., Tokyo ŽJapan., respectively. MX was synthesized in the Laboratory of Food Hygiene, School of Food and Nutritional Sciences, University of Shizuoka ŽJapan.. MMC, DDP, and BLM were dissolved in Hanks’ balanced salt solution ŽpH 7.0., and the other compounds were dissolved in dimethyl sulfoxide ŽDMSO, Wako Pure Chemical Industries, Ltd... Regular ŽGP42. and low melting point ŽLGT. agarose were obtained from Nacalai Tesque, Inc., and were diluted to 0.75% in physiological saline. Chinese hamster ovary ŽCHO K1. cells, obtained originally from American Type Culture Collection and cloned, were grown in Ham’s F12 medium ŽNissui Pharmaceutical Inc., Tokyo, Japan. supplemented with 10% fetal bovine serum ŽHyClone Laboratories, Inc., USA.. Both cell lines were grown in a humidified atmosphere with 5% CO 2 in air at 378C. 2.2. Treatment Logarithmic growth phase cells Ž5 = 10 5 cellsrml treatment medium. were treated with each chemical at 378C for 1 h. The doses at which clastogenic effects were shown in CHO cells without cytotoxicity were chosen on the basis of our previous studies w7–9x. To investigate the induction of crosslinks and intercalation, CHO cells were treated with 5 mgrml BLM for 1 h followed by treatment for 1 h with MMC, DDP, or AMD. CHO cells were sampled immediately after chemical treatment and the percentage of viable cells was measured by the Trypan blue exclusion test. 2.3. Slide preparation Seventy five ml 0.75% agarose GP-42 was quickly layered on a fully frosted slide ŽMatsunami Glass Ind., Ltd., Osaka, Japan. and covered with another slide glass. The slides were placed on ice to allow the agarose to gel. Treated cells were suspended in 0.75% agarose-LGT and incubated at 378C and 75
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
ml of cell suspension Ž7.5 = 10 4 cellsr75 ml. was quickly layered at the same manner after removing the slide glass. Finally, 75 ml of 0.75% agarose GP-42 was quickly laid on again. The slides were lysed immediately in a solution ŽpH 10. of 2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100 at 48C for 60 min w1x. 2.4. Unwinding and electrophoresis The slides were placed on a horizontal gel electrophoresis platform, and covered with an alkaline solution made up of 300 mM NaOH and 1 mM Na 2 EDTA Žadjusted to pH 12.1 and 12.6 by HCl.. The slides were left in the solution for 20 min to allow the unwinding of the DNA and expression of alkali-labile sites. The power supply was set at 25 V and 250 mA. The DNA was electrophoresed for 20 min and the slides were rinsed gently 3 times with 400 mM Trizma ŽpH 7.5. to neutralize the excess alkali. Each slide was stained with 50 ml of 20 mgrml ethidium bromide ŽWako Pure Chemical Industries, Ltd.. and covered with a coverslip. 2.5. Examination of the cells Fifty cells on one slide per treatment group were examined and photographed Žblack and white 400 ASA Fuji film. in a fluorescence microscope ŽOlympus at 200 = magnification. equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. The frequency of cells with a tail Ž‘tailed cells’. was scored, and the whole length of the comet Ž‘length’. and the diameter of the head Ž‘diameter’. were measured. The shape factor was calculated as the ratio of length to diameter. Migration was calculated as the difference between length and diameter. 2.6. Statistical analysis The effect of chemical treatment on frequency of tailed cells, shape factor, and migration was analyzed using a one-tailed trend test. We used the cumulative x 2-test for frequency of tailed cells and Student’s t-test for shape factor and migration. The data at different pHs was compared statistically using slope comparison.
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3. Results The results of the alkaline SCG assay with CHO cells are shown in Table 1. Three independent experiments were conducted and representative data were shown. BLM and MX dose-dependently increased tailed cell frequency, shape factor, and DNA migration at both pH 12.1 and 12.6. In BLM-treated cells, the results were the same at pH 12.1 and 12.6. On the other hand, in MX-treated cells, the positive response at pH 12.6 was greater than it was at pH 12.1. MNU, ENU, MNNG, ENNG, MMS, EMS, and 4-NQO induced a positive response only at pH 12.6. MMC, DDP, and AMD showed no effects irrespective of pH. Table 2 shows the effect of MMC, DDP, and AMD on the positive response of BLM in CHO cells. BLM showed a positive response both at pH 12.1 and 12.6; MMC- and DDP-treatment suppressed the positive response dose-dependently. AMD did not affect BLM-induced tails. 4. Discussion In our study, BLM and MX induced positive responses at pH 12.1 and 12.6, while MNU, ENU, MNNG, ENNG, MMS, and 4-NQO induced positive response only at pH 12.6. These results suggest that SSBs will be detected at pH 12.1, and both SSBs and alkali-labile sites will be detected at pH G 12.6 in the SCG assay. This assay detects DNA lesions as SSB-shortened DNA molecules. BLM is an Sphase-independent, X-ray mimetic mutagen that induces SSBs w10x; the other tested compounds are S-phase-dependent, UV mimetic mutagens that induce alkylated bases and bulky base adducts, which develop into alkali-labile sites w2,11,12x. This would account for the positive responses induced by BLM at pH 12.1 and 12.6 and by the other mutagens at pH 12.6 only. MX, a mutagenic by-product of chlorination of tap water, showed interesting results. MX produces apurinicrapyrimidinic ŽAP. sites inducing alkali-labile sites in great excess of direct strand breaks w13x. We showed that MX gave positive responses both at pH 12.1 and pH 12.6, and that the latter response was greater. These results are in good agreement with the previous report w13x.
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
110
MMC and DDP are DNA crosslinkers w14–17x. Since DNA crosslinking lesions inhibit DNA unwinding, it would be expected that they inhibit the
fragmentation of DNA molecules rather than induce a comet tail and that incomplete repair sites of crosslinkings produce DNA migration. Our findings
Table 1 DNA damage detected at pH 12.1 and 12.6 in CHO cells after 1 h of mutagen treatment Mutagen
MX
Dose pH 12.1 ŽmM. Frequency Shape factor of tailed cells Ž%.
b
4.0 18.0 e 42.0 f 50.0 f 48.0 f p - 0.05
0 0.24 0.71 2.4 7.1
2.0 1.09 " 0.65 46.0 f 2.41 " 1.65 60.0 f 3.02 " 1.36 66.0 f 3.02 " 1.36 76.0 f 3.88 " 2.08 p - 0.001 p - 0.001
Trend test MNU
0 64.7 194 647 1940
Trend test ENU
NS
Trend test
a
2.0 1.12 " 0.82 70.0 f 4.31 " 2.63 88.0 f 4.48 " 2.82 92.0 f 3.32 " 1.54 98.0 f 5.49 " 2.86 p - 0.001 p - 0.001
3.79 " 26.6 51.3 " 57.4 f 60.0 " 73.8 f 72.3 " 44.5 f 101 "61.0 f p - 0.001
4.0 1.08 " 0.48 44.0 f 2.83 " 2.46 64.0 f 3.10 " 1.85 64.0 f 3.10 " 1.85 72.0 f 3.09 " 1.85 p - 0.001 p - 0.001
1.02 " 0.08 1.06 " 0.32 1.00 " 0.00 1.00 " 0.00 1.08 " 0.33 NS
0.82 " 3.89 3.34 " 17.0 0.00 " 0.00 0.00 " 0.00 4.10 " 17.6 NS
8.0 1.18 " 0.62 25.5 e 2.04 " 2.15 46.7 f 3.02 " 2.70 68.5 f 5.39 " 4.43 98.0 f 6.02 " 1.84 p - 0.001 p - 0.001
f f f f
Migration Žmm.
f f f f
f f f f
d e f f
1.03 " 1.17 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.06 " 0.26 NS
1.85 " 9.10 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 3.08 " 12.3 NS
6.5 1.56 " 2.25 84.3 f 5.01 " 2.72 86.7 f 4.91 " 2.55 100 f 3.81 " 1.45 100 f 6.45 " 3.18 p - 0.001 p - 0.001
0 0.023 0.07 0.23 0.70
0.0 0.0 0.0 2.0 2.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.02 " 0.14 1.04 " 0.20 NS
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 1.03 " 7.18 2.36 " 11.7 NS
4.7 1.18 " 0.82 73.7 f 4.32 " 3.29 92.0 f 4.39 " 1.85 86.0 f 4.36 " 2.14 95.7 f 4.90 " 1.58 p - 0.001 p - 0.001
0 2.07 6.2 20.7 62
6.0 8.0 0.0 0.0 4.0 NS
1.07 " 0.29 1.13 " 0.50 1.00 " 0.00 1.00 " 0.00 1.20 " 0.99 NS
3.90 " 15.6 6.97 " 26.1 0.00 " 0.00 0.00 " 0.00 6.67 " 32.7 NS
6.8 1.30 " 1.13 100 f 6.61 " 1.48 100 f 6.91 " 1.88 94.0 f 5.40 " 1.83 100 f 6.76 " 4.18 p - 0.001 p - 0.001
0 6.17 18.5 61.7
0.0 0.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 NS
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 NS
4.0 1.19 " 1.22 73.2 f 3.85 " 2.05 f 100 f 3.23 " 0.61 f f 74.0 2.56 " 1.17 f p - 0.001 p - 0.001
Trend test MMS
Frequency Shape factor of tailed cells Ž%.
4.0 0.0 0.0 0.0 6.0 NS
Trend test ENNG
c
a
0 45.7 137 457 1370
Trend test MNNG
6.0 4.0 0.0 0.0 6.0
Migration Žmm.
1.20 " 0.99 6.67 " 32.7 1.44 " 1.01 19.8 " 45.6 2.04 " 1.44 e 34.2 " 44.4 f 2.36 " 1.81 f 43.2 " 50.3 f e 2.67 " 2.78 49.2 " 60.0 f p - 0.05 p - 0.001
0 0.15 0.46 1.5 4.6
Trend test BLM
pH 12.6 a
f f f f
f f f f
f f f f
a
Viable cells Ž% of control.
3.59 " 25.1 100 71.3 " 49.0 f 95 71.3 " 31.7 f 104 65.6 " 27.6 f 91 113 "38.5 f 89 p - 0.001 3.69 " 20.3 100 60.0 " 79.0 f 114 76.5 " 66.7 f 102 76.5 " 66.7 f 91 66.7 " 54.9 f 91 p - 0.001 8.67 " 30.4 100 31.4 " 56.9 d 89 61.0 " 75.9 d 105 103 " 79.0 f 111 144 "39.5 f 88 p - 0.001 12.3 " 46.7 100 100 "55.9 f 91 98.5 " 57.4 f 90 78.5 " 24.1 f 100 114 "16.4 f 89 p - 0.001 5.49 " 24.6 100 82.6 " 68.2 f 91 94.9 " 45.7 f 88 91.8 " 46.0 f 111 81.5 " 32.9 f 102 p - 0.001 9.90 " 36.7 100 159 " 14.9 f 102 173 " 51.2 f 85 118 " 38.4 f 95 136 " 21.0 f 91 p - 0.001 3.28 " 19.0 100 88.2 " 59.5 f 92 86.2 " 16.6 f 103 50.6 " 33.9 f 95 p - 0.001
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
111
Table 1 Žcontinued. Mutagen
EMS
Dose ŽmM.
Shape factor
0.0 0.0 0.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 NS
0 0.14 0.42 1.4 4.2
6.0 0.0 2.0 0.0 0.0 NS
0 0.083 0.25 0.83 2.5
0 26.8 80.5 268 805
Trend test MMC
Trend test DDP
0 0.56 1.68 5.6 16.8
Trend test AMD
Trend test
pH 12.6 a
Frequency of tailed cells Ž%.
Trend test 4-NQO
pH 12.1
0 0.0013 0.004 0.013 0.04
Migration Žmm.
a
a
Migration Žmm.
Viable cells Ž% of control.
a
Frequency of tailed cells Ž%.
Shape factor
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 NS
2.0 56.0 f 51.2 f 62.8 f 70.0 f p - 0.001
1.07 " 0.51 2.68 " 1.85 f 2.58 " 1.95 f 3.38 " 2.82 f 3.69 " 2.21 f p - 0.001
1.07 " 0.29 1.00 " 0.00 1.08 " 0.44 1.00 " 0.00 1.00 " 0.00 NS
3.90 " 15.6 0.00 " 0.00 4.10 " 22.6 0.00 " 0.00 0.00 " 0.00 NS
8.0 26.0 e 28.0 e 96.0 f 92.0 f p - 0.001
1.48 " 1.60 2.57 " 3.36 2.26 " 2.18 4.82 " 2.77 6.08 " 2.60 p - 0.001
6.0 0.0 4.0 4.0 6.0 NS
1.16 " 0.64 1.00 " 0.00 1.10 " 0.49 1.08 " 0.40 1.06 " 0.24 NS
8.21 " 32.6 0.00 " 0.00 5.13 " 21.1 4.10 " 20.8 3.08 " 12.2 NS
6.0 2.0 2.0 4.0 8.0 NS
1.07 " 0.31 1.07 " 0.47 1.03 " 0.24 1.08 " 0.38 1.08 " 0.32 NS
2.87 " 12.9 2.77 " 19.4 1.95 " 13.6 3.18 " 15.7 3.38 " 13.3 NS
100 92 105 114 102
0.0 0.0 0.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 NS
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 NS
2.0 4.0 6.0 6.0 0.0 NS
1.03 " 0.21 1.07 " 0.37 1.08 " 0.39 1.12 " 0.52 1.00 " 0.00 NS
1.54 " 10.8 3.10 " 15.9 3.07 " 13.3 4.31 " 17.4 0.00 " 0.00 NS
100 105 95 121 89
0.0 0.0 2.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.05 " 0.35 1.00 " 0.00 1.00 " 0.00 NS
0.00 " 0.00 0.00 " 0.00 2.56 " 17.9 0.00 " 0.00 0.00 " 0.00 NS
6.0 2.0 10.0 6.0 6.0 NS
1.55 " 2.22 1.10 " 0.70 1.25 " 0.86 1.22 " 0.90 1.22 " 0.93 NS
10.9 " 43.0 2.05 " 14.4 6.87 " 21.3 5.85 " 23.4 5.74 " 24.0 NS
100 118 94 92 102
2.26 " 15.8 51.8 " 52.3 f 43.7 " 46.7 e 50.1 " 47.6 f 79.0 " 57.9 f p - 0.001
100 85 89 82 88
11.3 " 34.8 32.4 " 54.9 31.6 " 51.8 89.7 " 29.2 143 "54.8 p - 0.001
100 92 91 105 103
d f f
d f f
a
Values are the mean " the standard deviations of the mean. Data based on 50 cells per sample. Concentration was calculated by the molecular weight of bleomycin A 2 . c NS, not significant. d–f Significant difference from control ŽStudent’s t-test.: d 0.01 - p - 0.05; e 0.001 - p - 0.01; f p - 0.001. b
were that MMC and DDP were negative immediately after MMC treatment and reduced the BLM positive response. In another paper, we reported that MMC led to a positive response after the recovery period, but not just after treatment w18x. These results show that the alkaline SCG assay detects crosslinking lesions and incomplete repair sites in a predictable manner. On the other hand, AMD was not positive under the present experimental conditions.
The mutagenic mechanisms of AMD is intercalation and the compound does not react with DNA. Considering that the SCG assay detects mutagenesis as a lowering of DNA length, our present results are consistent with known mechanisms. The alkaline SCG assay is therefore useful for the detection of mutagens that induce SSBs, alkali-labile sites, or DNA crosslinks. Our results demonstrated that the alkaline condition is an important factor for
112
Table 2 The effect of MMC, DDP, and AMD on the positive response of BLM-treated CHO cells in the SCG assay Mutagen
0
y MMC
7.1
Trend test
y DDP
7.1
y AMD
7.1
Trend test
c
pH 12.6 a
Shape factor
y 2.5 0 0.083 0.25 0.83 2.5
4.0 8.0 62.0 58.0 48.0 e 34.0 g 30.0 g p- 0.001
1.04"0.21 1.06"0.22 2.73"1.51 1.97"0.97 1.90"1.15 1.54"0.93 1.52"0.77 p- 0.05
y 16.8 0 1.68 5.6 16.8
0.0 0.0 70.0 70.0 40.0 g 32.0 g p- 0.001
1.00"0.00 1.00"0.00 3.14"1.74 3.03"1.28 2.22"1.65 e 1.97"1.26 g p- 0.05
y 0.04 0 0.004 0.013 0.04
0.0 0.0 70.0 70.0 72.0 68.0 NS d
c
0
pH 12.1 Frequency of tailed cells Ž%.
c
0
Trend test
Dose ŽmM.
1.00"0.00 1.00"0.00 3.14"1.74 3.26"1.51 3.00"1.89 3.28"1.54 NS
f f g g
Migration a Žmm.
Frequency of tailed cells Ž%.
Shape factor
1.85"9.08 2.26"8.26 70.2"57.9 36.2"35.7 g 28.6"33.9 g 29.1"33.9 g 17.8"23.5 f p- 0.001
4.0 8.0 76.0 60.0 e 42.0 g 44.0 g 32.0 g p- 0.001
1.03"0.20 1.16"0.44 2.84"1.70 1.90"1.02 1.66"0.87 1.64"0.92 1.62"1.06 p- 0.05
0.00"0.00 0.00"0.00 66.3"44.2 72.4"25.7 40.2"52.4 39.2"44.7 f p- 0.05
0.0 0.0 68.0 66.7 38.0 g 36.0 g p- 0.001
1.00"0.00 1.00"0.00 3.09"1.66 2.87"1.38 2.23"1.69 2.06"1.27 p- 0.05
0.00"0.00 0.00"0.00 66.3"44.2 75.4"28.7 76.3"44.2 75.3"50.2 NS
0.0 0.0 68.0 76.0 68.0 70.0 NS
a
1.00"0.00 1.00"0.00 3.09"1.56 3.05"1.98 3.90"2.80 3.48"2.00 NS
CHO cells treated with 7.1 mM BLM for 1 h were treated with DNA crosslinking agents MMC, DDP, and AMD for 1 h. a Values are the mean"the standard deviations of the mean. Data based on 50 cells per sample. b Concentration was calculated by the molecular weight of bleomycin A 2 . c Decrease in migration was analyzed using a one-tailed trend test. d NS, not significant. e–g Significant decrease from cells treated with 7.1 mM BLM ŽStudent’s t-test.: e 0.01- p- 0.05; f 0.001- p- 0.01; g p- 0.001.
Migration a Žmm.
f f f f
e f
Viable cells Ž% of untreated.
1.03"7.18 5.85"16.4 55.4"48.3 30.4"31.5 f 25.6"36.5 f 23.4"29.4 g 15.6"23.9 g p- 0.001
100 92 102 101 105 92 89
0.00"0.00 0.00"0.00 62.1"44.3 66.0"39.4 40.2"52.8 43.2"48.5 e p- 0.05
100 105 102 87 94 100
0.00"0.00 0.00"0.00 62.1"44.3 70.1"45.9 70.5"59.0 73.9"53.4 NS
100 94 102 95 90 88
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
BLM b ŽmM.
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
the alkaline SCG assay to detect the genotoxic effects of chemicals. w8x
Acknowledgements This work was supported by a Grant-in-Aid from the Tutikawa Memorial Fund for Study in Mammalian Mutagenicity. We also thank Mr. Michiyoshi Muraki, president of Shirakaba Farm Co., Ltd. ŽKaduno, Akita, Japan., for his financial support. The authors are grateful to Dr. Miriam Bloom for her critical reading of the manuscript.
References w1x N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 Ž1988. 184–191. w2x D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutation Res. 339 Ž1995. 37–59. w3x E.C. Friedberg, DNA Repair, Freeman Co., New York, 1984. w4x 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. w5x S. Pfuhler, H.U. Wolf, Detection of DNA-crosslinking agents with the alkaline comet assay, Environ. Mol. Mutagen. 27 Ž1996. 196–201. w6x R. Vijayalaxmi, R. Tice, G.H.S. Strauss, Assessment of radiation-induced DNA damage in human lymphocytes using single cell gel electrophoresis technique, Mutation Res. 271 Ž1992. 243–252. w7x Y.F. Sasaki, H. Yamada, C. Sugiyama, N. Kinae, Increasing
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w18x
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effects of tri-n-butyltins and triphenyltins on the frequency of chemically induced chromosome aberrations in cultured Chinese hamster cells, Mutation Res. 300 Ž1993. 5–14. Y.F. Sasaki, M. Sakaguchi, T. Yamagishi, H. Yamada, Y. Shirasu, Bio-anticlastogenic effects of unsaturated fatty acids included in fish oil – docosahaxaenoic acid, docosapentaenoic acid, and eicosapentaenoic acid – in cultured Chinese hamster cells, Mutation Res. 320 Ž1994. 9–22. Y.F. Sasaki, N. Kinae, Co-clastogenic effects of water pollutants, Environ. Mutation Res. Commun. 17 Ž1995. 65–74. B.K. Vig, R. Lewis, Genetic toxicology of bleomycin, Mutation Res. 49 Ž1978. 189–238. E.C. Friedberg, U.K. Ehmann, J.I. Williams, Human diseases associated with defective DNA repair, Adv. Radiat. Biol. 8 Ž1979. 85–174. P.C. Hanawalt, P.K. Cooper, A.K. Ganesan, C.A. Smith, DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem. 48 Ž1979. 783–803. J.M.T. Hyttinen, K. Jansson, PM2 DNA damage induced by 3-chloro-4-Ždichloromethyl. -5-hydroxy-2-Ž 5 H .-furanone ŽMX., Mutation Res. 348 Ž1995. 183–186. W. Szybalski, Crosslinking of DNA by enzymatically or chemically activated mitomycins and porfiromycins, bifunctionally alkylating antibiotics, Fed. Proc. 23 Ž1964. 946–957. M.S. Sasaki, A. Tonomura, A high susceptibility in Fanconi’s anemia to chromosome breakage by DNA crosslinking agents, Cancer Res. 33 Ž1973. 1829–1836. M.S. Sasaki, Is Fanconi’s anemia defective in a process essential for the repair of DNA crosslinks?, Nature 257 Ž1975. 501–503. M.S. Sasaki, Fanconi’s anemia: a condition possibly associated with defective DNA repair. In: P.C. Hanawalt, E.C. Friedberg, C.F. Fox ŽEds.., DNA Repair Mechanisms, Academic Press, New York, 1978, pp. 675–684. Y. Miyamae, K. Zaizen, K. Ohara, Y. Mine, Y.F. Sasaki, Detection of DNA lesions induced by chemical mutagens by the single cell gel electrophoresis ŽComet. assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens, Mutation Res. 393 Ž1997. 99–106.
Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis ž Comet/ assay. 2. Relationship between DNA migration and alkaline condition Youichi Miyamae a , Kouichi Iwasaki b,1, Naohide Kinae b, Shuji Tsuda c , Michiko Murakami d,2 , Makiko Tanaka d,3, Yu F. Sasaki d,) a
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 1-6, 2-chome, Kashima, Yodogawa-Ku, Osaka 532, Japan Laboratory of Food Hygiene, School of Food and Nutritional Sciences, UniÕersity of Shizuoka, Yada 52-1, Shizuoka 422, Japan c Laboratory of Veterinary Public Health, Faculty of Agriculture, Iwate UniÕersity, Ueda 3-18-8, Morioka, Iwate 020, Japan Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 16-1, Hachinohe, Aomori 039-11, Japan b
d
Received 21 May 1996; revised 11 April 1997; accepted 23 April 1997
Abstract The alkaline condition is an important factor for the alkaline single-cell gel electrophoresis ŽSCG. assay to detect the genotoxic effects of chemicals. In order to understand the relationship between DNA migration and alkaline condition, the effect of 13 model chemical mutagens with different modes of action was evaluated with the alkaline SCG assay under two different alkaline conditions ŽpH 12.1 and 12.6.. CHO cells were sampled just after treatment for 1 h. The X-ray mimetic mutagen BLM increased DNA migration at pH 12.1 and 12.6 and the results were the same at both pH values. Six alkylating mutagens MNU, ENU, MNNG, ENNG, MMS, and EMS and one base adduct inducer 4-NQO induced a dose-dependent response only at pH 12.6. Two DNA crosslinking agents, MMC and DDP, and AMD had negative results. MMC and DDP, however, reduced the positive response of BLM, suggesting that DNA crosslinks could be detected. These results demonstrated that the alkaline condition was important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals. q 1997 Elsevier Science B.V. Keywords: Alkaline single cell gel electrophoresis ŽSCG. assay; Comet assay; DNA lesion; pH; Chemical mutagen
X
X
Abbreviations: MNNG, N-methyl-N -nitro-N-nitrosoguanidine; ENNG, N-ethyl-N -nitro-N-nitrosoguanidine; MNU, methyl nitrosourea; ENU, ethyl nitrosourea; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; 4-nitroquinoline 1-oxide, 4-NQO; MMC, mitomycin C; DDP, cis-diaminodichloroplatin; AMD, actinomycin D; BLM, bleomycin; MX, 3-chloro-4-dichloromethyl-5-hydroxy2Ž5H .-furanone; SSB, DNA single-strand break ) Corresponding author. Tel.rfax: q81 Ž178. 27-7296; e-mail: [email protected] 1 Present address: Water Research Foundation Shizuoka Prefecture, Kita-andoh, Shizuoka 420, Japan. 2 Present address: Faculty of Engineering, The Technology University of Nagaoka, Kamitomioka 1603-1, Nagaoka, Niigata 940-21, Japan. 3 Present address: Tokyo Factory, Nisshinbo Co. Ltd., Nishiarai Sakae 1-18-1, Adachi, Tokyo 123, Japan. 1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 9 1 - 0
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Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
1. Introduction The alkaline single-cell gel electrophoresis ŽSCG. assay is a rapid and sensitive procedure for quantitating DNA lesions in mammalian cells w1,2x. In this assay, cells are embedded in agarose, lysed in an alkaline buffer, and subjected to an electric current. Relaxed and broken DNA fragments stream further from the nucleus than intact DNA, so the extent of DNA damage can be measured by the length of the stream. DNA is denatured and unwound and electrophoresis is conducted under alkaline condition ŽpH ) 12. w1,2x. At pH G 12.6, SSBs develop from DNA lesions called alkali-labile sites w3x. Thus, only SSBs will be detected at pH 12.1, and both SSBs and alkali-labile sites will be detected at pH G 12.6. Since DNA crosslinking lesions which inhibit DNA unwinding inhibit the fragmentation of DNA molecules rather than induce a comet tail, DNA crosslinking lesions as well as SSBs and alkali-labile sites can be detected by the alkaline SCG assay w4,5x. Some kinds of mutagen react DNA with UVandror X-ray mimetic modeŽs. and induce SSBs andror alkali-labile sites. The alkaline condition, therefore, is an important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals. In this study, we questioned the relationship between DNA migration and alkaline condition. We selected the following as model mutagens: the S N -1 type alkylating agents MNNG, ENNG, MNU, and ENU, the S N -2 type alkylating agents MMS and EMS, the base adduct inducers 4-NQO and MX, the crosslinking agents MMC and DDP, the DNA intercalator AMD, the X-ray mimetic mutagen BLM. According to Vijayalaxmi, et al., pH ) 13 would be more useful than pH 12.6 for detecting certain classes of alkali-labile lesions w6x. To understand the relationship between DNA migration and alkaline condition, however, we used the lowest pH ŽpH 12.6. at which alkali-labile sites could be detected. 2. Materials and methods 2.1. Chemicals, cells and medium MNNG, ENNG, MMS, and EMS were obtained from Sigma Chemicals Inc., St. Louis, MO ŽUSA..
MNU and ENU were purchased from Nacalai Tesque, Inc., Kyoto ŽJapan.. 4-NQO was obtained from Wako Pure Chemical Industries, Ltd., Osaka ŽJapan.. MMC and DDP came from Kyowa Hakko Kogyo Ltd., Tokyo ŽJapan., and Nihon Kayaku Industries, Ltd., Tokyo ŽJapan., respectively. MX was synthesized in the Laboratory of Food Hygiene, School of Food and Nutritional Sciences, University of Shizuoka ŽJapan.. MMC, DDP, and BLM were dissolved in Hanks’ balanced salt solution ŽpH 7.0., and the other compounds were dissolved in dimethyl sulfoxide ŽDMSO, Wako Pure Chemical Industries, Ltd... Regular ŽGP42. and low melting point ŽLGT. agarose were obtained from Nacalai Tesque, Inc., and were diluted to 0.75% in physiological saline. Chinese hamster ovary ŽCHO K1. cells, obtained originally from American Type Culture Collection and cloned, were grown in Ham’s F12 medium ŽNissui Pharmaceutical Inc., Tokyo, Japan. supplemented with 10% fetal bovine serum ŽHyClone Laboratories, Inc., USA.. Both cell lines were grown in a humidified atmosphere with 5% CO 2 in air at 378C. 2.2. Treatment Logarithmic growth phase cells Ž5 = 10 5 cellsrml treatment medium. were treated with each chemical at 378C for 1 h. The doses at which clastogenic effects were shown in CHO cells without cytotoxicity were chosen on the basis of our previous studies w7–9x. To investigate the induction of crosslinks and intercalation, CHO cells were treated with 5 mgrml BLM for 1 h followed by treatment for 1 h with MMC, DDP, or AMD. CHO cells were sampled immediately after chemical treatment and the percentage of viable cells was measured by the Trypan blue exclusion test. 2.3. Slide preparation Seventy five ml 0.75% agarose GP-42 was quickly layered on a fully frosted slide ŽMatsunami Glass Ind., Ltd., Osaka, Japan. and covered with another slide glass. The slides were placed on ice to allow the agarose to gel. Treated cells were suspended in 0.75% agarose-LGT and incubated at 378C and 75
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
ml of cell suspension Ž7.5 = 10 4 cellsr75 ml. was quickly layered at the same manner after removing the slide glass. Finally, 75 ml of 0.75% agarose GP-42 was quickly laid on again. The slides were lysed immediately in a solution ŽpH 10. of 2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100 at 48C for 60 min w1x. 2.4. Unwinding and electrophoresis The slides were placed on a horizontal gel electrophoresis platform, and covered with an alkaline solution made up of 300 mM NaOH and 1 mM Na 2 EDTA Žadjusted to pH 12.1 and 12.6 by HCl.. The slides were left in the solution for 20 min to allow the unwinding of the DNA and expression of alkali-labile sites. The power supply was set at 25 V and 250 mA. The DNA was electrophoresed for 20 min and the slides were rinsed gently 3 times with 400 mM Trizma ŽpH 7.5. to neutralize the excess alkali. Each slide was stained with 50 ml of 20 mgrml ethidium bromide ŽWako Pure Chemical Industries, Ltd.. and covered with a coverslip. 2.5. Examination of the cells Fifty cells on one slide per treatment group were examined and photographed Žblack and white 400 ASA Fuji film. in a fluorescence microscope ŽOlympus at 200 = magnification. equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. The frequency of cells with a tail Ž‘tailed cells’. was scored, and the whole length of the comet Ž‘length’. and the diameter of the head Ž‘diameter’. were measured. The shape factor was calculated as the ratio of length to diameter. Migration was calculated as the difference between length and diameter. 2.6. Statistical analysis The effect of chemical treatment on frequency of tailed cells, shape factor, and migration was analyzed using a one-tailed trend test. We used the cumulative x 2-test for frequency of tailed cells and Student’s t-test for shape factor and migration. The data at different pHs was compared statistically using slope comparison.
109
3. Results The results of the alkaline SCG assay with CHO cells are shown in Table 1. Three independent experiments were conducted and representative data were shown. BLM and MX dose-dependently increased tailed cell frequency, shape factor, and DNA migration at both pH 12.1 and 12.6. In BLM-treated cells, the results were the same at pH 12.1 and 12.6. On the other hand, in MX-treated cells, the positive response at pH 12.6 was greater than it was at pH 12.1. MNU, ENU, MNNG, ENNG, MMS, EMS, and 4-NQO induced a positive response only at pH 12.6. MMC, DDP, and AMD showed no effects irrespective of pH. Table 2 shows the effect of MMC, DDP, and AMD on the positive response of BLM in CHO cells. BLM showed a positive response both at pH 12.1 and 12.6; MMC- and DDP-treatment suppressed the positive response dose-dependently. AMD did not affect BLM-induced tails. 4. Discussion In our study, BLM and MX induced positive responses at pH 12.1 and 12.6, while MNU, ENU, MNNG, ENNG, MMS, and 4-NQO induced positive response only at pH 12.6. These results suggest that SSBs will be detected at pH 12.1, and both SSBs and alkali-labile sites will be detected at pH G 12.6 in the SCG assay. This assay detects DNA lesions as SSB-shortened DNA molecules. BLM is an Sphase-independent, X-ray mimetic mutagen that induces SSBs w10x; the other tested compounds are S-phase-dependent, UV mimetic mutagens that induce alkylated bases and bulky base adducts, which develop into alkali-labile sites w2,11,12x. This would account for the positive responses induced by BLM at pH 12.1 and 12.6 and by the other mutagens at pH 12.6 only. MX, a mutagenic by-product of chlorination of tap water, showed interesting results. MX produces apurinicrapyrimidinic ŽAP. sites inducing alkali-labile sites in great excess of direct strand breaks w13x. We showed that MX gave positive responses both at pH 12.1 and pH 12.6, and that the latter response was greater. These results are in good agreement with the previous report w13x.
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
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MMC and DDP are DNA crosslinkers w14–17x. Since DNA crosslinking lesions inhibit DNA unwinding, it would be expected that they inhibit the
fragmentation of DNA molecules rather than induce a comet tail and that incomplete repair sites of crosslinkings produce DNA migration. Our findings
Table 1 DNA damage detected at pH 12.1 and 12.6 in CHO cells after 1 h of mutagen treatment Mutagen
MX
Dose pH 12.1 ŽmM. Frequency Shape factor of tailed cells Ž%.
b
4.0 18.0 e 42.0 f 50.0 f 48.0 f p - 0.05
0 0.24 0.71 2.4 7.1
2.0 1.09 " 0.65 46.0 f 2.41 " 1.65 60.0 f 3.02 " 1.36 66.0 f 3.02 " 1.36 76.0 f 3.88 " 2.08 p - 0.001 p - 0.001
Trend test MNU
0 64.7 194 647 1940
Trend test ENU
NS
Trend test
a
2.0 1.12 " 0.82 70.0 f 4.31 " 2.63 88.0 f 4.48 " 2.82 92.0 f 3.32 " 1.54 98.0 f 5.49 " 2.86 p - 0.001 p - 0.001
3.79 " 26.6 51.3 " 57.4 f 60.0 " 73.8 f 72.3 " 44.5 f 101 "61.0 f p - 0.001
4.0 1.08 " 0.48 44.0 f 2.83 " 2.46 64.0 f 3.10 " 1.85 64.0 f 3.10 " 1.85 72.0 f 3.09 " 1.85 p - 0.001 p - 0.001
1.02 " 0.08 1.06 " 0.32 1.00 " 0.00 1.00 " 0.00 1.08 " 0.33 NS
0.82 " 3.89 3.34 " 17.0 0.00 " 0.00 0.00 " 0.00 4.10 " 17.6 NS
8.0 1.18 " 0.62 25.5 e 2.04 " 2.15 46.7 f 3.02 " 2.70 68.5 f 5.39 " 4.43 98.0 f 6.02 " 1.84 p - 0.001 p - 0.001
f f f f
Migration Žmm.
f f f f
f f f f
d e f f
1.03 " 1.17 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.06 " 0.26 NS
1.85 " 9.10 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 3.08 " 12.3 NS
6.5 1.56 " 2.25 84.3 f 5.01 " 2.72 86.7 f 4.91 " 2.55 100 f 3.81 " 1.45 100 f 6.45 " 3.18 p - 0.001 p - 0.001
0 0.023 0.07 0.23 0.70
0.0 0.0 0.0 2.0 2.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.02 " 0.14 1.04 " 0.20 NS
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 1.03 " 7.18 2.36 " 11.7 NS
4.7 1.18 " 0.82 73.7 f 4.32 " 3.29 92.0 f 4.39 " 1.85 86.0 f 4.36 " 2.14 95.7 f 4.90 " 1.58 p - 0.001 p - 0.001
0 2.07 6.2 20.7 62
6.0 8.0 0.0 0.0 4.0 NS
1.07 " 0.29 1.13 " 0.50 1.00 " 0.00 1.00 " 0.00 1.20 " 0.99 NS
3.90 " 15.6 6.97 " 26.1 0.00 " 0.00 0.00 " 0.00 6.67 " 32.7 NS
6.8 1.30 " 1.13 100 f 6.61 " 1.48 100 f 6.91 " 1.88 94.0 f 5.40 " 1.83 100 f 6.76 " 4.18 p - 0.001 p - 0.001
0 6.17 18.5 61.7
0.0 0.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 NS
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 NS
4.0 1.19 " 1.22 73.2 f 3.85 " 2.05 f 100 f 3.23 " 0.61 f f 74.0 2.56 " 1.17 f p - 0.001 p - 0.001
Trend test MMS
Frequency Shape factor of tailed cells Ž%.
4.0 0.0 0.0 0.0 6.0 NS
Trend test ENNG
c
a
0 45.7 137 457 1370
Trend test MNNG
6.0 4.0 0.0 0.0 6.0
Migration Žmm.
1.20 " 0.99 6.67 " 32.7 1.44 " 1.01 19.8 " 45.6 2.04 " 1.44 e 34.2 " 44.4 f 2.36 " 1.81 f 43.2 " 50.3 f e 2.67 " 2.78 49.2 " 60.0 f p - 0.05 p - 0.001
0 0.15 0.46 1.5 4.6
Trend test BLM
pH 12.6 a
f f f f
f f f f
f f f f
a
Viable cells Ž% of control.
3.59 " 25.1 100 71.3 " 49.0 f 95 71.3 " 31.7 f 104 65.6 " 27.6 f 91 113 "38.5 f 89 p - 0.001 3.69 " 20.3 100 60.0 " 79.0 f 114 76.5 " 66.7 f 102 76.5 " 66.7 f 91 66.7 " 54.9 f 91 p - 0.001 8.67 " 30.4 100 31.4 " 56.9 d 89 61.0 " 75.9 d 105 103 " 79.0 f 111 144 "39.5 f 88 p - 0.001 12.3 " 46.7 100 100 "55.9 f 91 98.5 " 57.4 f 90 78.5 " 24.1 f 100 114 "16.4 f 89 p - 0.001 5.49 " 24.6 100 82.6 " 68.2 f 91 94.9 " 45.7 f 88 91.8 " 46.0 f 111 81.5 " 32.9 f 102 p - 0.001 9.90 " 36.7 100 159 " 14.9 f 102 173 " 51.2 f 85 118 " 38.4 f 95 136 " 21.0 f 91 p - 0.001 3.28 " 19.0 100 88.2 " 59.5 f 92 86.2 " 16.6 f 103 50.6 " 33.9 f 95 p - 0.001
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
111
Table 1 Žcontinued. Mutagen
EMS
Dose ŽmM.
Shape factor
0.0 0.0 0.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 NS
0 0.14 0.42 1.4 4.2
6.0 0.0 2.0 0.0 0.0 NS
0 0.083 0.25 0.83 2.5
0 26.8 80.5 268 805
Trend test MMC
Trend test DDP
0 0.56 1.68 5.6 16.8
Trend test AMD
Trend test
pH 12.6 a
Frequency of tailed cells Ž%.
Trend test 4-NQO
pH 12.1
0 0.0013 0.004 0.013 0.04
Migration Žmm.
a
a
Migration Žmm.
Viable cells Ž% of control.
a
Frequency of tailed cells Ž%.
Shape factor
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 NS
2.0 56.0 f 51.2 f 62.8 f 70.0 f p - 0.001
1.07 " 0.51 2.68 " 1.85 f 2.58 " 1.95 f 3.38 " 2.82 f 3.69 " 2.21 f p - 0.001
1.07 " 0.29 1.00 " 0.00 1.08 " 0.44 1.00 " 0.00 1.00 " 0.00 NS
3.90 " 15.6 0.00 " 0.00 4.10 " 22.6 0.00 " 0.00 0.00 " 0.00 NS
8.0 26.0 e 28.0 e 96.0 f 92.0 f p - 0.001
1.48 " 1.60 2.57 " 3.36 2.26 " 2.18 4.82 " 2.77 6.08 " 2.60 p - 0.001
6.0 0.0 4.0 4.0 6.0 NS
1.16 " 0.64 1.00 " 0.00 1.10 " 0.49 1.08 " 0.40 1.06 " 0.24 NS
8.21 " 32.6 0.00 " 0.00 5.13 " 21.1 4.10 " 20.8 3.08 " 12.2 NS
6.0 2.0 2.0 4.0 8.0 NS
1.07 " 0.31 1.07 " 0.47 1.03 " 0.24 1.08 " 0.38 1.08 " 0.32 NS
2.87 " 12.9 2.77 " 19.4 1.95 " 13.6 3.18 " 15.7 3.38 " 13.3 NS
100 92 105 114 102
0.0 0.0 0.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 1.00 " 0.00 NS
0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 0.00 " 0.00 NS
2.0 4.0 6.0 6.0 0.0 NS
1.03 " 0.21 1.07 " 0.37 1.08 " 0.39 1.12 " 0.52 1.00 " 0.00 NS
1.54 " 10.8 3.10 " 15.9 3.07 " 13.3 4.31 " 17.4 0.00 " 0.00 NS
100 105 95 121 89
0.0 0.0 2.0 0.0 0.0 NS
1.00 " 0.00 1.00 " 0.00 1.05 " 0.35 1.00 " 0.00 1.00 " 0.00 NS
0.00 " 0.00 0.00 " 0.00 2.56 " 17.9 0.00 " 0.00 0.00 " 0.00 NS
6.0 2.0 10.0 6.0 6.0 NS
1.55 " 2.22 1.10 " 0.70 1.25 " 0.86 1.22 " 0.90 1.22 " 0.93 NS
10.9 " 43.0 2.05 " 14.4 6.87 " 21.3 5.85 " 23.4 5.74 " 24.0 NS
100 118 94 92 102
2.26 " 15.8 51.8 " 52.3 f 43.7 " 46.7 e 50.1 " 47.6 f 79.0 " 57.9 f p - 0.001
100 85 89 82 88
11.3 " 34.8 32.4 " 54.9 31.6 " 51.8 89.7 " 29.2 143 "54.8 p - 0.001
100 92 91 105 103
d f f
d f f
a
Values are the mean " the standard deviations of the mean. Data based on 50 cells per sample. Concentration was calculated by the molecular weight of bleomycin A 2 . c NS, not significant. d–f Significant difference from control ŽStudent’s t-test.: d 0.01 - p - 0.05; e 0.001 - p - 0.01; f p - 0.001. b
were that MMC and DDP were negative immediately after MMC treatment and reduced the BLM positive response. In another paper, we reported that MMC led to a positive response after the recovery period, but not just after treatment w18x. These results show that the alkaline SCG assay detects crosslinking lesions and incomplete repair sites in a predictable manner. On the other hand, AMD was not positive under the present experimental conditions.
The mutagenic mechanisms of AMD is intercalation and the compound does not react with DNA. Considering that the SCG assay detects mutagenesis as a lowering of DNA length, our present results are consistent with known mechanisms. The alkaline SCG assay is therefore useful for the detection of mutagens that induce SSBs, alkali-labile sites, or DNA crosslinks. Our results demonstrated that the alkaline condition is an important factor for
112
Table 2 The effect of MMC, DDP, and AMD on the positive response of BLM-treated CHO cells in the SCG assay Mutagen
0
y MMC
7.1
Trend test
y DDP
7.1
y AMD
7.1
Trend test
c
pH 12.6 a
Shape factor
y 2.5 0 0.083 0.25 0.83 2.5
4.0 8.0 62.0 58.0 48.0 e 34.0 g 30.0 g p- 0.001
1.04"0.21 1.06"0.22 2.73"1.51 1.97"0.97 1.90"1.15 1.54"0.93 1.52"0.77 p- 0.05
y 16.8 0 1.68 5.6 16.8
0.0 0.0 70.0 70.0 40.0 g 32.0 g p- 0.001
1.00"0.00 1.00"0.00 3.14"1.74 3.03"1.28 2.22"1.65 e 1.97"1.26 g p- 0.05
y 0.04 0 0.004 0.013 0.04
0.0 0.0 70.0 70.0 72.0 68.0 NS d
c
0
pH 12.1 Frequency of tailed cells Ž%.
c
0
Trend test
Dose ŽmM.
1.00"0.00 1.00"0.00 3.14"1.74 3.26"1.51 3.00"1.89 3.28"1.54 NS
f f g g
Migration a Žmm.
Frequency of tailed cells Ž%.
Shape factor
1.85"9.08 2.26"8.26 70.2"57.9 36.2"35.7 g 28.6"33.9 g 29.1"33.9 g 17.8"23.5 f p- 0.001
4.0 8.0 76.0 60.0 e 42.0 g 44.0 g 32.0 g p- 0.001
1.03"0.20 1.16"0.44 2.84"1.70 1.90"1.02 1.66"0.87 1.64"0.92 1.62"1.06 p- 0.05
0.00"0.00 0.00"0.00 66.3"44.2 72.4"25.7 40.2"52.4 39.2"44.7 f p- 0.05
0.0 0.0 68.0 66.7 38.0 g 36.0 g p- 0.001
1.00"0.00 1.00"0.00 3.09"1.66 2.87"1.38 2.23"1.69 2.06"1.27 p- 0.05
0.00"0.00 0.00"0.00 66.3"44.2 75.4"28.7 76.3"44.2 75.3"50.2 NS
0.0 0.0 68.0 76.0 68.0 70.0 NS
a
1.00"0.00 1.00"0.00 3.09"1.56 3.05"1.98 3.90"2.80 3.48"2.00 NS
CHO cells treated with 7.1 mM BLM for 1 h were treated with DNA crosslinking agents MMC, DDP, and AMD for 1 h. a Values are the mean"the standard deviations of the mean. Data based on 50 cells per sample. b Concentration was calculated by the molecular weight of bleomycin A 2 . c Decrease in migration was analyzed using a one-tailed trend test. d NS, not significant. e–g Significant decrease from cells treated with 7.1 mM BLM ŽStudent’s t-test.: e 0.01- p- 0.05; f 0.001- p- 0.01; g p- 0.001.
Migration a Žmm.
f f f f
e f
Viable cells Ž% of untreated.
1.03"7.18 5.85"16.4 55.4"48.3 30.4"31.5 f 25.6"36.5 f 23.4"29.4 g 15.6"23.9 g p- 0.001
100 92 102 101 105 92 89
0.00"0.00 0.00"0.00 62.1"44.3 66.0"39.4 40.2"52.8 43.2"48.5 e p- 0.05
100 105 102 87 94 100
0.00"0.00 0.00"0.00 62.1"44.3 70.1"45.9 70.5"59.0 73.9"53.4 NS
100 94 102 95 90 88
Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
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Y. Miyamae et al.r Mutation Research 393 (1997) 107–113
the alkaline SCG assay to detect the genotoxic effects of chemicals. w8x
Acknowledgements This work was supported by a Grant-in-Aid from the Tutikawa Memorial Fund for Study in Mammalian Mutagenicity. We also thank Mr. Michiyoshi Muraki, president of Shirakaba Farm Co., Ltd. ŽKaduno, Akita, Japan., for his financial support. The authors are grateful to Dr. Miriam Bloom for her critical reading of the manuscript.
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