Use of single cell gel electrophoresis (comet assay) modifications for analysis of DNA damage.
Single cell gel electrophoresis (SCGE) or comet assay is a rapid and sensitive fluorescent microscopic method which allows measurement of DNA strand breaks in individual cells. Modifications of SCGE conditions permitted to detect different types of DNA damage. In order to characterize DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2) in Chinese hamster V79 cells, two approaches were used: (1) two pH values of unwinding and electrophoresis solutions (pH > or = 13.0 and pH = 12.1) to specify the type of DNA lesions [the alkali-labile sites and true DNA single-strand breaks (ssb)] and (2) DNA glycosylases [endonuclease III (EndoIII) and formamidopyrimidine-DNA glycosylase (FaPy)] or DNA inhibitors [hydroxyurea (HU) + 1-(beta-D-arabinofuranosyl)cytosine (AraC)] to characterize the types of DNA damage. Our results showed that the lesions induced by H2O2 represented mainly the true DNA ssb, while MNNG formed predominantly alkali-labile sites, which were converted to DNA ssb under strong alkaline conditions (pH > or = 13.0). The effects of DNA repair enzymes and DNA inhibitors were more significant under lower pH (pH = 12.1) of unwinding and electrophoresis solution. Both, DNA glycosylases and DNA inhibitors increased the level of DNA ssb.
Horváthová
E
E
Cancer Research Institute, Slovak Academy of Sciences, Department of Mutagenesis and Carcinogenesis, Bratislava. exonhore@savba.sk
Slamenová
D
D
Gábelová
A
A
D000818:Animals; D002460:Cell Line; D020552:Comet Assay / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003561:Cytarabine / Q000633:toxicity*; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000187:drug effects; D043382:DNA-Formamidopyrimidine Glycosylase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006918:Hydroxyurea / Q000633:toxicity*; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D003561:Cytarabine; D008769:Methylnitronitrosoguanidine; D006861:Hydrogen Peroxide; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D006918:Hydroxyurea