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 microM or of MMC at 20, 40, and 60 microM 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.
Hincks
J R
JR
Graduate Programs in Toxicology, Utah State University, Logan.
Coulombe
R A
RA
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000302:isolation & purification* / Q000528:radiation effects; D004249:DNA Damage; D005374:Filtration / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008466:Mechlorethamine / Q000494:pharmacology; D016685:Mitomycin; D008937:Mitomycins / Q000494:pharmacology; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D008937:Mitomycins; D008466:Mechlorethamine; D016685:Mitomycin; D004247:DNA