Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification.
The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.
Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Kifissia, GR-145 61 Athens, Greece.
Marina
M
Goumenou
Kyriaki
K
Machera
D000818:Animals; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000032:analysis / Q000737:chemistry* / Q000187:drug effects; D005374:Filtration / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D009153:Mutagens / Q000494:pharmacology; D009691:Nucleic Acid Denaturation; D051381:Rats; D017208:Rats, Wistar
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009153:Mutagens