Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (Comet) assay.: 2. Relationship between DNA migration and alkaline condition

doi:10.1016/S1383-5718(97)00091-0

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Volume 393, Issues 1–2, 18 September 1997, Pages 107-113

https://doi.org/10.1016/S1383-5718(97)00091-0 Get rights and content

Abstract

The alkaline condition is an important factor for the alkaline single-cell gel electrophoresis (SCG) 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 (pH 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.

Introduction

The alkaline single-cell gel electrophoresis (SCG) assay is a rapid and sensitive procedure for quantitating DNA lesions in mammalian cells 1, 2. 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 (pH >12) 1, 2. At pH ≥12.6, SSBs develop from DNA lesions called alkali-labile sites [3]. Thus, only SSBs will be detected at pH 12.1, and both SSBs and alkali-labile sites will be detected at pH ≥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 4, 5.

Some kinds of mutagen react DNA with UV- and/or X-ray mimetic mode(s) and induce SSBs and/or 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 [6]. To understand the relationship between DNA migration and alkaline condition, however, we used the lowest pH (pH 12.6) at which alkali-labile sites could be detected.

Section snippets

Chemicals, cells and medium

MNNG, ENNG, MMS, and EMS were obtained from Sigma Chemicals Inc., St. Louis, MO (USA). MNU and ENU were purchased from Nacalai Tesque, Inc., Kyoto (Japan). 4-NQO was obtained from Wako Pure Chemical Industries, Ltd., Osaka (Japan). MMC and DDP came from Kyowa Hakko Kogyo Ltd., Tokyo (Japan), and Nihon Kayaku Industries, Ltd., Tokyo (Japan), respectively. MX was synthesized in the Laboratory of Food Hygiene, School of Food and Nutritional Sciences, University of Shizuoka (Japan). MMC, DDP, and

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

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 ≥12.6 in the SCG assay. This assay detects DNA lesions as SSB-shortened DNA molecules. BLM is an S-phase-independent, X-ray mimetic mutagen that induces SSBs [10]; the other tested compounds are S-phase-dependent, UV

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. (Kaduno, 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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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.

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Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (Comet) assay.: 2. Relationship between DNA migration and alkaline condition

Abstract

Introduction

Section snippets

Chemicals, cells and medium

Results

Discussion

Acknowledgements

Exp. Cell. Res.

Mutation Res.

Exp. Cell Res.

Mutation Res.

Mutation Res.

Adv. Radiat. Biol.

Mutation Res.

Mutation Res.