A FLUORESCENCE ENHANCEMENT ASSAY FOR CELLULAR DNA DAMAGE

  • 1 January 1982
    • journal article
    • research article
    • Vol. 22  (1) , 145-151
Abstract
A fluorescence procedure is described for quantitative measurement of DNA damage in mammalian cells. The technique is based on the time-dependent partial alkaline unwinding of cellular DNA followed by determination of duplex:total DNA ratios with bisbenzamide, which has a differential molar fluorescence with single-stranded and duplex DNA. The method is rapid, does not require radioactive labeling of DNA and is sufficiently sensitive to detect damage induced with 100 rad of X-irradiation. This method is standardized with respect to the alkaline unwinding unit, Mn0, and the unwinding constant, .beta.. Results obtained with this new technique and with hydroxylapatite chromatography for physical separation of single- and double-stranded DNA were confirmatory. The utility of the technique was demonstrated by detection of dose-related damage with X-irradiation and a variety of antineoplastic agents [actinomycin D, 1,3-bis(2-chloroethyl)-1-nitrosourea, daunorubicin, adriamycin, carminomycin, bleomycin, hycanthone, neocarzinostatin and 4''-(9-acridinylamino)-methansulfon-m-aniside] in unlabeled murine leukemia [P-388 and L-1210] cells.