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How to deal with your hedgehog comets

When performing the comet assay, comets which appear to have no head and a large bright tail may arise. These atypical comets are referred to as ‘hedgehogs’ or ‘clouds’ or ‘ghosts’. Recently, Lorenzo et al. have reviewed the incidence and perception of hedgehogs in the comet assay. 

There is speculation as to what these hedgehogs are. Historically, it was widely assumed that they represented apoptotic cells and, if believed, then the amount of hedgehogs observed can be used as an indicator for cytotoxicity. However, this theory is widely contested because the amount of DNA damage seen within a hedgehog is far less than expected for an apoptotic cell. Furthermore, hedgehogs have been observed after the moderate exposure of cells to a genotoxic agent, but when the cells were incubated for a short period, hedgehogs are no longer seen. This suggests that DNA repair is taking place in the affected cells.

It is possible that in some cases hedgehogs represent cells in the early stages of apoptosis. When cells were incubated with a cytotoxic (rather than genotoxic) agent, comets were observed. However, it is important to note that apoptosis is an irreversible process. If you need to investigate the apoptotic effect of a compound on cells, then you should employ a specific apoptotic assay.

Whatever you believe hedgehogs to be, experts suggest that the number of hedgehogs observed is recorded. Hedgehog data should not be ignored or disregarded, especially if the amount of hedgehogs seen in one sample is significantly higher than previously observed.

Within Comet Assay IV, there is a dedicated Hedgehog Counter. The counter allows the user to record the presence of a hedgehog with a single click. The user can chose whether to score the hedgehog or not, and a tally count of all the observed hedgehogs can be recorded.

 

Further reading:

The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead. Lorenzo Y, Costa S, Collins AR, Azqueta A. Mutagenesis. 2013 Jul;28(4):427-32.

Comet Assay measurements: a perspective. Kumaravel TS, Vilhar B, Faux SP, Jha AN. Cell Biol Toxicol. 2009 Feb;25(1):53-64.

Ecotoxicological applications and significance of the comet assay. Jha AN. Mutagenesis. 2008 May;23(3):207-21.

The comet assay: topical issues. Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, Smith CC, Stetina R. Mutagenesis. 2008 May;23(3):143-51.

Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A; In Vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing. Mutat Res. 2007 Feb 3;627(1):31-5.

The comet assay: genotoxic damage or nuclear fragmentation? Rundell MS, Wagner ED, Plewa MJ. Environ Mol Mutagen. 2003;42(2):61-7.

Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis. Meintières S, Nesslany F, Pallardy M, Marzin D. Environ Mol Mutagen. 2003;41(4):260-9.

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