The comet assay: Genotoxic damage or nuclear fragmentation?

Abstract

The single cell gel electrophoresis (SCGE) or comet assay is based on the assumption that comet images result from genotoxic damage that ultimately generate DNA single- or double-strand breaks. A criticism of the assay is that some or all of the comet images may be the result of apoptosis-mediated nuclear fragmentation. The objective of this study was to determine if mutagen-induced DNA damage leading to strand breakage observed in the SCGE assay was repairable or was due to nonrepairable nuclear fragmentation. Chinese hamster ovary cells were treated with ethylmethanesulfonate, 2-acetoxyacetylaminofluorene, or H(2)O(2). These mutagens induce genetic damage by different molecular mechanisms. One group of SCGE slides was prepared immediately after treatment, while parallel treated cultures were repeatedly washed and allowed to undergo liquid holding recovery for DNA repair. It was hypothesized that cells with genotoxic damage can repair their genomic DNA, while apoptotic cells cannot reverse nuclear fragmentation. We found a significant decrease in the tail moments of nuclei from mutagen-treated cells after 4 hr of liquid holding. However, this measurement may represent only those cells capable of repair. Apoptotic cells may continue DNA fragmentation during the recovery time and this DNA may become so diffuse that the nuclei disappear after electrophoresis. To overcome this possible artifact, images of nuclei were captured before and after alkaline electrophoresis. Constellations of nuclei were located on SCGE slides by their coordinates on the microscope stage. We found that no nuclei were lost due to apoptotic nuclear fragmentation and DNA migration. Even the so-called "hedgehog" comet images with extreme DNA damage were not lost during liquid holding. These data support the conclusion that mutagen-induced DNA damage is the principal cause of the damage measured in the comet assay.

Hydrogen Sulfide Induces Direct Radical-Associated DNA Damage

“…In the present study, we employed the alkaline single-cell gel electrophoresis (SCGE) assay or comet assay that quantitatively measures genomic DNA damage as singleand double-strand breaks or lesions that can be converted to strand breaks (28,29). SCGE is a sensitive genotoxicity assay and it has a high correlation in predicting carcinogens (30).…”

Double-Strand Break Repair in Plants Is Developmentally Regulated

“…This method is very sensitive when used under alkaline conditions and detects SSBs, DSBs, and other lesions that relax DNA (Cotelle and Ferard, 1999;Collins, 2004). To be specific to DSBs, this method should be used under a neutral pH, where the sensitivity of this method drops significantly (Rundell et al, 2003;Collins, 2004). However, several papers suggest the existence of false-positive results in apoptotic cells, potentially confounding the results of an experiment such as ours that focuses on the developmental stages of plants (Choucroun et al, 2001;Czene et al, 2002).…”

“…It has been suggested that an efficient method for detection of strand breaks is the Comet assay (Rundell et al, 2003;Collins, 2004). This method is based on the detection of gel shifts between damaged and nondamaged DNA due to a difference in supercoiling and relies on software-based measurements of the tail length of relaxed DNA molecules (Rundell et al, 2003;Collins, 2004).…”

“…This method is based on the detection of gel shifts between damaged and nondamaged DNA due to a difference in supercoiling and relies on software-based measurements of the tail length of relaxed DNA molecules (Rundell et al, 2003;Collins, 2004). This method is very sensitive when used under alkaline conditions and detects SSBs, DSBs, and other lesions that relax DNA (Cotelle and Ferard, 1999;Collins, 2004).…”

“…However, several papers suggest the existence of false-positive results in apoptotic cells, potentially confounding the results of an experiment such as ours that focuses on the developmental stages of plants (Choucroun et al, 2001;Czene et al, 2002). Since the method that we used is based on the direct incorporation of radionucleotides into 3#OH ends (see ''Materials and Methods''), we warranted it more appropriate than the Comet assay for our experiments (Rundell et al, 2003;Collins, 2004).…”