Gichner, Tomáš; Mukherjee, A.; Wagner, E. D.; Plewa, M. J.
MUTATION RESEARCH - GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 586 [1]: 38-46, 2005

Keywords: DNase-I; Ethyl methanesulphonate; Hydrogen peroxide
Abstract: We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apo-ptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA da-mage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) fol-lowed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bro-mide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treat-ments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants. (c) 2005 Elsevier B.V. All rights reserved.
DOI: IEB authors: Tomáš Gichner