Detection of DNA strand breaks and oxidized DNA bases at the single-cell level resulting from exposure to estradiol and hydroxylated metabolites

Both X-rays and the radiomimetic agent bleomycin (BLM) induce DNA strand breaks, predominantly via reactive radicals. To compare the induction of breaks with the two agents in Chinese hamster (CHO-K1) cells, two different alkaline unwinding methods, a 3H tracer-based analysis of large cell populations and an optical adaption allowing measurement of single cells, were applied. Radiation and BLM show qualitatively similar dose responses when the average number of DNA strand breaks is measured in a large cell population. However, the breakage pattern at the single-cell level indicates large discrepancies between the actions of the two agents. Irradiated cells show a uniform distribution of DNA strand breaks over the cell population. Effects of treatment with 30 micrograms x ml-1 BLM for 2 hr vary from practically zero in some cells to high levels of DNA strand breakage in others. Unlike the repair of radiation-induced DNA breaks, the repair efficiency of BLM-induced DNA strand breaks, as measured at the single-cell level, varies strongly among cells of the same population. Such heterogeneity at the cellular level potentially reduces BLM's usefulness for tumor therapy because the appearance of BLM-resistant subpopulations may critically impair treatment outcome.

Download Full-text

The fast halo assay: An improved method to quantify genomic DNA strand breakage at the single-cell level

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2006.04.018 ◽

2006 ◽

Vol 607 (2) ◽

pp. 205-214 ◽

Cited By ~ 42

Author(s):

Piero Sestili ◽

Chiara Martinelli ◽

Vilberto Stocchi

Keyword(s):

Single Cell ◽

Genomic Dna ◽

Single Cell Level ◽

Improved Method ◽

Cell Level ◽

Strand Breakage ◽

Dna Strand ◽

Dna Strand Breakage

Download Full-text

Determining DNA strand breaks using the laser scanning microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014720x ◽

1993 ◽

Vol 51 ◽

pp. 272-273

Author(s):

M.K. Winters ◽

D.W. Fairbairn ◽

M.D. Standing ◽

K.L. O’Neill

Keyword(s):

Dna Damage ◽

Single Cell ◽

Ethidium Bromide ◽

Laser Scanning ◽

Accurate Method ◽

Dna Strand Breaks ◽

Scanning Microscope ◽

Strand Breaks ◽

Dna Breakage ◽

Dna Strand

The single cell gel assay has been proven to be an effective, fast and accurate method of determining the amount of DNA single or double stranded breaks. The possible uses of this assay have already reached into some aspects of DNA damage caused by chemicals or other destructive agents. It has yet to reach even deeper into mutagenesis and carcinogenesis, both of which may show single or double stranded breaks to the DNA. Here we explore the effects that different concentrations of H2O2 have on the DNA of cells. The assay is performed by suspending cells in a low melt point agarose, spreading the agarose out on a frosted slide and letting it gel over ice. The cells are then treated with H2O2 to induce single strand DNA breakage. The cells are lysed in an alkaline environment, which separates the DNA into single strands and then electrophoresed. The gel is then stained with ethidium bromide.

Download Full-text