Statistics of the Comet assay: a key to discriminate between genotoxic effects

Abstract

The alkaline Comet assay is a widely used single cell gel electrophoresis technique for the quantification of DNA strand breaks, crosslinks and alkali-labile sites induced by a series of physical and chemical agents. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA, often reported parameters being tail DNA and tail moment. Although a variety of statistical approaches have been used in the literature, most of these do not take into account the distribution patterns of comet data. In order to investigate a methodology for statistically demonstrating a comet effect, two different experiments, a reproducibility study and a trend analysis, were undertaken on a murine lymphoma cell line (P388D1) photodynamically stressed after induction of porphyrins with delta-aminolaevulinic acid. This treatment results in significant heterogeneity of DNA damage, producing values ranging from 0 to 100% tail DNA in the same sample. The comparison of distribution curves for stressed and non-stressed samples shows that none of the application conditions are verified, either for parametric tests (which require normal distributions), or non-parametric tests (which assume essentially similar distributions). Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Whether the considered comet parameter is tail DNA or tail moment had no influence on the conclusions of our experiments, which were carried up to stress levels leading to a median 70% tail DNA.

Potential Role of Mosquito Larvae <i>Culex Pipiens</i> as a Biological Indicator of Environmental Water Pollution in Egypt

“…White and Rasmussen, (1998) showed that TM has the advantage of considering damage expressed as a long tail with a low fraction of high fraction of DNA or a short tail with a highly fragmented DNA. There are another study revealed that tail length and tail moment can provide similar results (Duez et al, 2003).…”

“…Furthermore, TM has the upside of considering damage communicated as a short tail with a high division of DNA or a long tail with a low division of DNA (White and Rasmussen, 1998). According to Duez et al (2003) and Pereira et al (2010), the lower significance increase in TM data of the 4 th instar, pupae and male stages Elmansoreyah water stream may reflect the cells with DNA cross-linking lesions. From the above results, the tail moment more responsive than tail length, and these results agreed with data obtained by Pereira et al (2010).…”

The importance of iron chelation and iron availability during PpIX-induced photodynamic therapy

“…Data were presented as box-and-whisker plots where the median value was illustrated as a line flanked by boxes within which 25% to 75% of the data fell and whiskers indicating 10% to 90% of the data range observed. In a cell culture study Duez et al [55] concluded that analysis of median comet data from repeated experiments is an efficient way to demonstrate genotoxic effects with ALA [55].…”

Genotoxicity of food preservative sodium sorbate in human lymphocytes in vitro

“…DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA (Duez et al 2003). The Comet assay is a rapid, simple and sensitive technique for measuring DNA breakage with a small number of cells and detects intercellular differences in DNA damage Johanson 1984, 1987;Singh et al 1988).…”