A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Abstract

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H₂O₂. After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H₂O₂ concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.

Key words::

• chicken erythrocytes
• chromatin dispersion
• DNA fragmentation
• hydrogen peroxide
• ionizing radiation
• nuclear halos

Acknowledgments

We thank M. Valenzuela, D. Villagra, A. Vilaxa, J. Cáceres, P. González, and K. Soto for valuable collaboration. This work was supported by grants from the Dirección de Investigación de la Universidad de Tarapacá, Arica, Chile, 2010 (Research Project UTA-Mayor No. 7721-10), and Ministerio de Ciencia e Innovación (CTQ2010-20870-C03-03). A. Blázquez-Castro acknowledges the financial support (FPU Fellowship) from Ministerio de Ciencia e Innovación, Spain.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.