5.1 A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand breaks may be introduced in many ways, directly by genotoxic compounds, through the induction of apoptosis or necrosis, secondarily through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes (20-22). In addition to a linkage with cancer, studies have demonstrated that increases in cellular DNA damage precede or correspond with reduced growth, abnormal development, and reduced survival of adults, embryos, and larvae (16, 23, 24).

5.1.1 The Comet assay can be easily utilized for collecting data on DNA strand breakage (9, 25, 26). It is a simple, rapid, and sensitive method that allows the comparison of DNA strand damage in different cell populations. As presented in this guide, the assay facilitates the detection of DNA single strand breaks and alkaline labile sites in individual cells, and can determine their abundance relative to control or reference cells (9, 16, 26). The assay offers a number of advantages; damage to the DNA in individual cells is measured, only extremely small numbers of cells need to be sampled to perform the assay (<10 000), the assay can be performed on practically any eukaryotic cell type, and it has been shown in comparative studies to be a very sensitive method for detecting DNA damage (2, 27) .

5.1.2 These are general guidelines. There are numerous procedural variants of this assay. The variation used is dependent upon the type of cells being examined, the types of DNA damage of interest, and the imaging and analysis capabilities of the lab conducting the assay. To visualize the DNA, it is stained with a fluorescent dye, or for light microscope analysis the DNA can be silver stained (28). Only fluorescent staining methods will be described in this guide. The microscopic determination of DNA migration can be made either by eye using an ocular micrometer or with the use of image analysis software. Scoring by eye can be performed using a calibrated ocular micrometer or by categorizing cells into four to five classes based on the extent of migration (29, 30) . Image analysis systems are comprised of a CCD camera attached to a fluorescent microscope and software and hardware designed specifically to capture and analyze images of fluorescently stained nuclei. Using such a system, it is possible to measure the fluorescence intensity and distribution of DNA in and away from the nucleus (8). Using different procedural variants, the assay can be utilized to measure specific types of DNA alterations and DNA repair activity (1, 3, 8, 10, 13, 14, 17, 18). Alkaline lysis and electrophoresis conditions are used for the detection of single-stranded DNA damage, whereas neutral pH conditions facilitate the detection of double-strand breaks (31). Various sample treatments can be used to express specific types of DNA damage, or as in one method, to preserve strand damage at sites of DNA repair (10). Nuclease digestion steps can be used to introduce strand breaks at specific lesion sites. Using this approach, oxidative base damage can be detected by the use of endonuclease III (18), as well as DNA modifications resulting from exposure to ultraviolet light (UV) through the use of T4 endonuclease V (3). Modifications of this type vastly expand the utility of this assay and are good examples of its versatility.

5.2 A sufficient knowledge of the biology of cells examined using this assay should be attained to understand factors affecting DNA strand breakage and the distribution of this damage within sampled cell populations. This includes, but is not limited to, influences such as cell type heterogeneity, cell cycle, cell turnover frequency, culture or growth conditions, and other factors that may influence levels of DNA strand damage. Different cell types may have vastly different background levels of DNA single-strand breaks due to variations in excision repair activity, metabolic activity, anti-oxidant concentrations, or other factors. It is recommended that cells representing those to be studied using the SCG/Comet assay be examined under the light or fluorescent microscope using stains capable of differentially staining different cell types. Morphological differences, staining characteristics, and frequencies of the different cell types should be noted and compared to SCG/Comet damage profiles to identify any possible cell type specific differences. In most cases, the use of homogenous cell populations reduces inter-cell variability of SCG/Comet values. The procedures for this assay, using cells from many different species and cell types, have been published previously (1, 2, 3, 5, 8, 10, 13, 14, 17, 18, 32-38). These references and others should be consulted to obtain details on the collection, handling, storage, and preparation of specific cell types.

5.3 The experimental design should incorporate appropriate controls, reference samples, and replicates to delineate the influence of the major sources of experimental variability.

1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4).2 There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.

1.1.1 The Comet assay has been used to screen the genotoxicity of a variety of compounds on cells in vitro and in vivo (5-7), as well as to evaluate the dose-dependent anti-oxidant (protective) properties of various compounds (3, 8-11). Using this method, significantly elevated levels of DNA damage have been reported in cells collected from organisms at polluted sites compared to reference sites (12-15). Studies have also found that increases in cellular DNA damage correspond with higher order effects such as decreased growth, survival, and development, and correlate with significant increases in contaminant body burdens (13, 16).

1.2 This guide presents protocols that facilitate the expression of DNA alkaline labile single-strand breaks and the determination of their abundance relative to control or reference cells. The guide is a general one meant to familiarize lab personnel with the basic requirements and considerations necessary to perform the Comet assay. It does not contain procedures for available variants of this assay, which allow the determination of non-alkaline labile single-strand breaks or double-stranded DNA strand breaks (8), distinction between different cell types (13), identification of cells undergoing apoptosis (programmed cell death, (1, 17)), measurement of cellular DNA repair rates (10), detection of the presence of photoactive DNA damaging compounds (14), or detection of specific DNA lesions (3, 18) .

1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.4 This guide is arranged as follows:

Section

Scope

1

Referenced Documents

2

Terminology

3

Summary of Guide

4

Significance and Use

5

Equipment and Reagents

6

Assay Procedures

7

Treatment of Data

8

Reporting Data

9

Keywords

10

Annex

Annex A1

References

1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.