Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution.
Environ Mol Mutagen
; 13(3): 211-7, 1989.
Article
em En
| MEDLINE
| ID: mdl-2495939
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dano ao DNA
/
DNA
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Environ Mol Mutagen
Assunto da revista:
BIOLOGIA MOLECULAR
/
SAUDE AMBIENTAL
Ano de publicação:
1989
Tipo de documento:
Article