ATLA::Alternatives to Laboratory Animals

Volume 24, Number 3

Detection of oxidised purines and UV-induced photoproducts in DNA of single cells, by inclusion of lesion-specific enzymes in the comet assay.

ATLA 24, 405-411, May/June 1996

Mária Dusinská^1,2 and Andrew Collins³

¹Department of Mutagenesis and Chemical Carcinogenesis, Cancer Research Institute Spitalska 21, 81232 Bratislava, Slovakia; ³Rowett Research Institute, Greenhurn Road, Bucksourn, Aberdeen AB2 9SB, UK

SUMMARY

The comet assay (since cell gel electrophoresis) is a rapid, very sensitive method for the detection of DNA strand breaks at the level of single cells, which is now being applied in genotoxicity testing. We modified this method for the detection of a variety of kinds of DNA lesion, by treating nucleoid DNA in the gel with either formamidopyrimidine-DNA glycosylase (which recognizes ring opened purines, 8-hydroxyguanine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC; which has a rather broad specificity, including bulky lesions and W photoproducts). By using this modified assay, we demonstrate the removal of DNA strand breaks and oxidised purines upon incubating cells after treatment with hydrogen peroxide. This modification clearly increases the usefulness of the assay for the analysis of DNA damage and repair, for screening human populations for DNA damage, and for testing novel chemicals for genotoxicity.

Keywords: DNA damage, comet assay, formamidopyrimidine-DNA glycosylase, uvrABC excinuclease