Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution

Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross‐links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen‐induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA‐interstrand and DNA‐protein associated cross‐links in cultured epithelial cells. Cells were exposed to three known DNA cross‐linking agents, nitrogen mustard (HN2), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN2 at 0.25, 1.0, and 4.0 μM or of MMC at 20, 40, and 60 μM produced a dose‐dependent induction of total DNA cross‐links by these agents. Digestion with proteinase K revealed that HN2 and MMC induced both DNA‐protein cross‐links and DNA‐interstrand cross‐links. Ultraviolet irradiation induced both DNA cross‐links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity‐flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross‐linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents.