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

✍ Scribed by Jeffrey R. Hincks; Roger A. Coulombe Jr.

• Publisher: John Wiley and Sons
• Year: 1989
• Tongue: English
• Weight: 616 KB
• Volume: 13
• Category: Article
• ISSN: 0893-6692
• DOI: 10.1002/em.2850130304

✦ Synopsis

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 DNAinterstrand and DNA-protein associated crosslinks 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,l . O, and 4.0 JLM or of MMC at 20, 40, and 60 JLM produced a dosedependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN2 and MMC induced both DNAprotein cross-links and DNA-interstrand crosslinks. 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 anolyzed. 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.