A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.
In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.
Grigaravicius
Paulius
P
Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany. paulius@fli-leibniz.de
Rapp
Alexander
A
Greulich
Karl Otto
KO
D052160:Benzothiazoles; D001761:Bleomycin / Q000494:pharmacology*; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects / Q000528:radiation effects; D003851:Deoxyribonucleases / Q000378:metabolism*; D003959:Diamines; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D009930:Organic Chemicals; D011804:Quinolines; D011839:Radiation, Ionizing; D013194:Staining and Labeling; D014466:Ultraviolet Rays
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D001761:Bleomycin; C098022:SYBR Green I; D003851:Deoxyribonucleases