Quantitation of chemically induced DNA strand breaks in human cells via an alkaline unwinding assay.
DNA strand breaks induced in human CCRF-CEM cells by electrophilic chemicals (carcinogens/mutagens) can be readily quantitated via a facile alkaline unwinding assay. This procedure estimates the number of chemically induced DNA strand breaks on the basis of the percentage DNA converted from double-stranded to single-stranded form during an exposure to the alkaline unwinding conditions. The assay is based on the assumption that each strand break serves as a strand unwinding point during the alkaline denaturation. The extent of strand separation can be standardized with respect to the initial level of induced strand breaks by the use of X-rays, which produce known levels of DNA strand breaks per rad in mammalian cells. Subsequent to the alkaline exposure, the single- and double-stranded DNA were separated by use of thermostated hydroxylapatite columns (60 degrees C), and the DNA was quantitated via a fluorescence assay (Hoechst 33258 compound). A correlation was shown between mammalian DNA strand-breaking potential (as measured in this procedure) and the propensity of these chemicals to revert Salmonella typhimurium TA100.
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D002478:Cells, Cultured; D002586:Cesium; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides; D004247:DNA / Q000032:analysis*; D004260:DNA Repair / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000032:analysis; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D009153:Mutagens; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002712:Chlorides; D004277:DNA, Single-Stranded; D006882:Hydroxyapatites; D009153:Mutagens; D002586:Cesium; D004247:DNA; D017886:Durapatite; C028019:cesium chloride