Abstract

Immunoreactivity to fluorescein-labelled antinucleoside antibodies was induced in HeLa cell nuclei by the carcinogen N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). DNA strand breaks induced by MNNG were detected by measuring the influence of MNNG on the sedimentation in alkaline sucrose gradients of a fast sedimenting DNA-Containing complex. Dissociation of the complex and induction of immunoreactivity both proved to be sensitive, dose-dependent indicators of MNNG action in HeLa cells. Since antibodies directed against nucleosides react only with denatured or single-stranded DNA, it appears likely that MNNG-induced immunoreactivity results from exposure of single-stranded regions of DNA, providing a means for the rapid identification of carcinogen action by immunofluorescent techniques. N-Acetoxy-2-acetylaminofluorene also induced immunoreactivity and led to the dissociation of the DNA complex in HeLa cells. Seven other chemicals with known carcinogenic properties also induced immunoreactivity in this test. Immunoreactivity and dissociation of the complex were not induced by inhibitors of DNA synthesis such as hydroxyurea and cytosine arabinoside. Protein synthesis inhibitors such as cycloheximide induced immunoreactivity but did not dissociate the complex. Cycloheximide-induced immunoreactivity was rapidly and completely reversible following removal of the drug; the effects of MNNG were not reversible in our experiments. This novel application of immunofluorescent techniques provides a rapid quantitative means for detecting and studying the action of carcinogens in animal cells.

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